In the field of proteomics, efforts are devoted toward identifying in cell extracts the proteins present and the respective concentrations thereof, and also toward identifying the post-translational modifications of these proteins, which are representative of their activity. One of the post-translational modifications that is of greatest interest in this context is phosphorylation: protein phosphorylation is fundamental from a cognitive viewpoint in the field of modern biology, or from a diagnostic viewpoint in the therapeutic field. This function is in fact essential in many biological processes, such as epigenetic regulation, nutritional regulation, DNA repair, hormone regulation, etc. No known simple and direct technique makes it possible to identify a protein in a biological medium and to evaluate its degree of phosphorylation.
One known analytical technique consists in analyzing protein extracts by taking them up on “spot” sites (each spot consisting of a plurality of identical probes) organized in an array on a support, commonly referred to as a biochip or microarray. The targets sought in the protein extracts to be analyzed are identified with specific probes that may be antibodies, receptors or any other molecule that specifically associates with a given protein or with one of its modifications. In the case of antibodies, the support is termed an “antibody biochip”. A plurality of target biomolecules may thus be analyzed simultaneously or virtually simultaneously via suitable analytical techniques. Various techniques make it possible to perform the actual analysis of the elements thus retained or “segregated” on an antibody biochip.
For analysis of the phosphorylation of nucleic acids on a biochip, one known technique consists in performing this analysis via a technique of optical emission spectroscopy on a laser-induced plasma, commonly referred to by the abbreviation LIBS corresponding to the term laser-induced breakdown spectroscopy. This technique consists in ablating, by means of a laser beam, the segregated sample at the surface of the biochip and in generating a plasma, this plasma then being analyzed via a spectroscopic method. For each laser firing, the emission spectrum of the chemical elements investigated may be recorded, along with the focusing coordinates of the laser beam at the surface of the sample. Suitable calculation means may then make it possible to establish an elementary map of the surface of the sample. A device and a process for the quantitative measurement of biomolecular targets present on a biological analysis support are described, for example, in the patent application published under the reference FR 2 929 011. A LIBS analytical device is described, for example, in the patent application published under the reference FR 2 964 458.
However, a LIBS analysis of protein phosphorylation on a biochip poses several problems in principle. A first problem is associated with the presence of exogenous phosphorus, masking the signal sought. A second problem is associated with the fact that the supports usually used for biological analyses are incompatible with a LIBS analysis, since the plasmas which form on this type of support cannot be analyzed under acceptable conditions. A third problem is associated with the fact that biological molecules such as proteins have a very small amount of phosphorus in their structure, which is thus difficult to detect via LIBS. A fourth problem is associated with the fact that the signal obtained does not vary in a readily modelizable manner, for example linearly, as a function of the amount sought: in other words, normalization of the signal for the quantification of a protein is problematic.
One aim of the present invention is to overcome at least the abovementioned drawbacks, by proposing a process and a device for the quantitative analysis of biomolecular targets via a LIBS technique. This invention uses a suitable support and a process allowing the formation of a plasma that is readily analyzable by spectrometry, the LIBS signal emitted by the plasma being proportional to the abundance of the amount sought.
To this end, one subject of the invention is a process and a device for quantitative measurement. More specifically, one subject of the present invention is a process for the quantitative analysis by optical emission spectroscopy of a plasma induced by a laser beam of at least one target included in a biochip, characterized in that it involves the use of an adjuvant allowing the formation of a dry matrix capable of being ablated simultaneously with said at least one target, the dry matrix being configured to improve the analytical properties of plasma, the adjuvant having an emission spectrum whose lines have wavelengths distinct from the wavelengths of the spectral lines used for the quantitative analysis of said at least one target.
In one embodiment of the invention, said at least one target may be segregated on the biochip via at least one probe so that a probe-target complex is formed between each probe and the target corresponding thereto.
In one embodiment of the invention, said use of an adjuvant may allow the formation of a dry matrix of amorphous structure.
In one embodiment of the invention, said use of an adjuvant may allow the formation of a dry matrix of crystalline structure.
In one embodiment of the invention, the dry matrix may be formed such that the probe-target complexes are available at the surface of a support, and are totally or partially englobed by a volume of said dry matrix.
In one embodiment of the invention, the dry matrix and the probes may be fixed to a support.
In one embodiment of the invention, the adjuvant molecules forming the dry matrix may be directly complexed to the probes.
In one embodiment of the invention, the adjuvant molecules forming the dry matrix may be fixed to a support, the probes being fixed to the adjuvant molecules, the adjuvant molecules being fixed to the support.
In one embodiment of the invention, a layer of dry matrix may be formed by placing a layer of adjuvant on the surface of the support, prior to placing the probes on the surface of dry matrix thus formed at the surface of a support.
In one embodiment of the invention, the dry matrix may be formed by a compound whose absorption wavelengths are close to the wavelength of the laser beam, such that the wavelength used for the laser is included in the absorption spectrum of the dry matrix.
In one embodiment of the invention, the adjuvant may comprise a solution of water and of an element from the group comprising sugar, polysaccharide and disaccharide.
In one embodiment of the invention, each probe may be marked with a normalization element forming an internal standard.
In one embodiment of the invention, each probe may be marked with the normalization element by grafting.
In one embodiment of the invention, the normalization element may be formed by boron.
A subject of the present invention is also a device comprising a support comprising a plurality of sites, each site comprising a plurality of probes, the probes being grafted concomitantly to the molecules of an adjuvant allowing the formation of a dry matrix.
In one embodiment of the invention, said support may comprise organic compounds, monolithic compounds or organo-diamond compounds.
A subject of the present invention is also a solution for performing a process according to any one of the described embodiments, characterized in that it comprises an adjuvant allowing the formation of a dry matrix.
In one embodiment of the invention, the solution for performing a process according to one of the described embodiments may comprise a given proportion of an element for normalizing an optical signal of a plasma induced by a laser beam.
Another advantage afforded by the present invention is that it allows simple, rapid, quantitative and vectorial analysis of biomolecules, for example proteins, and of the phosphorus content in a mixture.
According to the present invention, a quantitative analysis is performed on a biochip comprising an array of targets and/or probes organized in spots. The spots of each probe are capable of recognizing and segregating a target biomolecule present in the mixture to be analyzed. According to one specificity of the present invention, it is proposed that an adjuvant be used to allow the formation of a dry matrix, this matrix being intimately linked with the targets or with the probe-target complexes. The term “intimately linked” means here that the dry matrix supports, englobes or surrounds the targets or the probe-target complexes, in a manner such that the laser ablation of the target or of the probe-target complex and the plasma produced by LIBS at a spot of the biochip array comprises, in addition to the target biomolecules or the target-probe complexes, adjuvant molecules and/or decomposition elements thereof. Hereinbelow, it is considered that a dry matrix is in the form of a solid compound of crystalline or amorphous form after drying of the biochip. As a nonlimiting example, the dry matrix may be in the form of an adduct, i.e. a mixture of probes, targets and adjuvant molecules, of a mixed crystalline or amorphous phase or englobing the targets and/or the probes. Various configurations of the dry matrix on the biochip support are described below as examples, with reference to FIGS. 2A to 2D. It should be observed that a process or a device according to the present invention may also apply to a biochip comprising a plurality of targets deposited or segregated on a support, not necessarily associated with probes.
The adjuvant is notably chosen so as to be essentially non-phosphorized, and so that it does not denature the antigen-antibody bond. This means, firstly, that the emission spectra of the molecules constituting the adjuvant and the matrix itself are different from the emission lines used to quantify the phosphorus and, where appropriate, to normalize the signal, and secondly that the adjuvant is compatible with the hybridization or complexation of the targets and/or of the probe-target complexes.
The adjuvant used for the formation of the dry matrix may, for example, be soluble. Examples of suitable solutions are described below. The dry matrix is also chosen so as moreover to have the advantage of improving the analytical properties of the plasma produced by the laser for the analysis via a spectroscopy technique. Notably, the dry matrix may be formed by a compound whose absorption wavelengths are close to the wavelength of the laser beam used, such that the wavelength used for the laser is included in the absorption spectrum of the dry matrix.